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A differential evolution algorithm was used to optimize the multi-variable, DMSO-free preservation protocol in 8 experiments. hiPSC aggregates frozen in the optimized solution did not exhibit the same sensitivity to undercooling as those frozen in non-optimized solutions or DMSO, indicating superior adaptability of the optimized solution to different freezing modalities and unplanned deviations. This investigation shows the importance of optimization, explains the mechanisms and advantages of a DMSO-free solution, and enables not only

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